The present invention relates to an immunoassay technique and in particular relates to a novel immunoassay method for quantitatively measuring a concentration of an antigen or an antibody in a sample.
There was reported by Yalow et al. (Nature, 184; 1948) a technique of measuring a slight amount of insulin by use of a specific antibody labelled with radioisotope. Since then, the measuring technique called radioimmunoassay has been utilized for the quantitative measurement of various biological materials and drugs. However, a problem at issue is the requiring of particular attention in handling, because of using a radioisotope. Thus, there have been investigated various immunoassay techniques utilizing nonisotopic labels such as enzyme, enzyme substrate, fluorescent and chemiluminescent materials. Above all, a technique of utilizing enzyme or fluorescent material as a label has reached a practical level. However, these techniques and the radioimmunoassay are still difficult to be automated, because of the labor and the time required for the entire process.
As a simplified technique for the entire process, one example that may be mentioned is that disclosed in U.S. Pat. No. 3,852,157 but in this patent an analyte is limited to haptens having a low molecular weight. This example shows the simplification of process without requiring an operation of separating an antigen-antibody complex from free antigens or antibodies. On the other hand, it has also been attempted to simplify the separation operation. The operation is called a solid phase method in which an antibody or antigen is bonded in advance on an insoluble carrier and the antigen-antibody reaction is caused on the carrier. The free antigens or antibodies are readily separable from the bound materials by washing the carrier with water.
The immunoassay process can be simplified by employing the solid phase method but many unsatisfactory points still remain in the aspect of the automation of the entire process. It is essential to shorten the time required for the entire process for automating the immunoassay. The antigen-antibody reaction requires time most in the immunoassay process and it usually takes several hours to one day therefor. It is usual that the required time becomes longer with the reduction of a concentration of material to be subject to the immunoassay.
For shortening the reaction time, there has been proposed a method of filling a column with microcrystals or fine particles bonded with an antibody (or antigen) and forcibly passing an analyte therethrough (Japanese Patent Publication No. 127823/78). This example shows a method of forcibly feeding a fluid itself such as the analyte into a portion or layer of a carrier in which an antibody or the like was immobilized.
As a method of feeding not a fluid wholly but only a specific component contained therein (e.g. a substance to be detected or the like), there is a method of selectively feeding a specific component by electrophoresis. In an example thereof, a portion of a gel layer disposed on a supporting plate is replaced with an antibody- or antigen-immobilized gel or porous matrix layer, and the antigen or antibody which is a material to be detected is fed into said portion (U.S. Pat. No. 3,966,897). In this case, the direction of feeding the material to be detected is necessarily parallel to a supporting plate surface, i.e. an antibody-immobilized layer surface.
Another example of employing the electrophoresis is shown in Japanese Patent Publication No. 132946/80. In this example, a gel tube filled with polyacrylamide for disk electrophoresis is prepared, and an antibody solution or antigen solution is poured into one end of the gel tube followed by electrophoresis thereby to form a concentrated layer of the antibody or antigen, and then the antigen-antibody reaction takes place in said concentrated layer while the antigen solution or antibody solution is being electrophoreted. It has also been tried to promote the formation of the concentrated layer by inserting a protein-impermeable membrane, e.g. a dialysis membrane, into a portion of the gel. Therein, the antibody or antigen is not immobilized in the supporting matrix for electrophoresis and is present in a free state in the reaction layer. Furthermore, because of being present in the free state in the reaction layer, the reaction layer itself cannot be directly contacted with an electrolyte solution in an anode side or in a cathode side of the electrophoresis.
In any of the antigen-antibody reaction methods utilizing the above cited two electrophoresis methods, the supporting matrix for electrophoresis (i.e. a portion free from the antibody or antigen) other than the reaction portion or reaction layer should necessarily be prepared. Thus, it is necessary to feed the antigen or antibody to the reaction portion or reaction layer through the supporting matrix for electrophoresis and accordingly, the distance of electrophoresis becomes longer and the time required for electrophoresis becomes longer. Furthermore, for effecting the immunoassay by employing these methods, an inhibitor e.g. an excess labelled antibody which does not participate in the reaction) fed to the reaction portion or reaction layer should be removed from the reaction portion or reaction layer. For this removal in the electrophoresis method, the electrophoresis should be further continued and in this case, more time is required for moving the inhibitor through the supporting matrix for electrophoresis other than the reaction portion or reaction layer.